Prolyl Hydroxylase Inhibitors: A Breakthrough in the Therapy of

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Cite This: J. Med. Chem. 2018, 61, 6964−6982

Prolyl Hydroxylase Inhibitors: A Breakthrough in the Therapy of Anemia Associated with Chronic Diseases Amit A. Joharapurkar,* Vrajesh B. Pandya, Vishal J. Patel, Ranjit C. Desai, and Mukul R. Jain

J. Med. Chem. 2018.61:6964-6982. Downloaded from pubs.acs.org by KAOHSIUNG MEDICAL UNIV on 08/23/18. For personal use only.

Zydus Research Centre, Cadila Healthcare Limited, Sarkhej Bavla NH8A, Moraiya, Ahmedabad 382210, India ABSTRACT: Chronic kidney disease, cancer, chronic inflammatory disorders, nutritional, and genetic deficiency can cause anemia. Hypoxia causes induction of hypoxia-inducible factor (HIF), which stimulates erythropoietin (EPO) synthesis. Prolyl hydroxylase domain (PHD) enzyme inhibition can stabilize hypoxia-inducible factor (HIF). HIF stabilization also decreases hepcidin, a hormone of hepatic origin, which regulates iron homeostasis. PHD inhibitors represent a novel pharmacological treatment of anemia associated with chronic diseases. Many orally active PHD inhibitors like roxadustat, molidustat, vadadustat, and desidustat are in late phase clinical trials. This review discusses the role of PHD inhibitors in the treatment of anemia associated with chronic diseases.

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which enucleate to form reticulocytes.11 In the bone marrow, CFU-Es and proerythroblasts are stimulated by EPO for further differentiation (Figure 1). Formation of erythrocytes from reticulocytes occurs within 24−48 h, and the mature erythrocytes circulate in the blood for 120 days, before phagocytosis in the spleen, liver, and bone marrow degrades them.12 EPO-mediated signaling is necessary for the survival and differentiation of erythroid progenitors. EPO binds to specific EPO receptors (EPORs) and signals through Janus tyrosine kinase-2 (JAK2).11 JAK2 stimulates signal transduction and activator of transcription-5 (STAT-5), RAS−RAF−MAP kinase, and phosphoinositide-3 kinase/AKT kinase (protein kinase B).13,14 On the other hand, erythroid progenitors undergo apoptosis by the cluster of differentiation 95 (CD95), a membrane protein of the tumor necrosis factor (TNF) receptor family, which triggers apoptosis after binding to the CD95 ligand, which is produced by mature erythroblasts. Thus, the negative feedback cycle controls the production of mature erythrocytes in blood.15 Iron is vital for hemoglobin synthesis and reticulocytes maturation. Hepcidin is a hepatic hormone that regulates iron metabolism.16 Ferroportin (FPN) is the ferrous exporter present in macrophages (reticuloendothelial cells), enterocytes, and hepatocytes. Hepcidin binds to ferroportin and causes its degradation by internalization and lysosomal digestion. This action of hepcidin causes iron entrapment in macrophages or enterocytes, creating a functional iron deficiency in the body. Erythropoiesis and hypoxia also regulate hepcidin levels.8 Increased iron levels (as in iron loading with iron supplementation) increases hepcidin production via bone

INTRODUCTION Anemia is a disorder of blood involving a decreased amount of red blood cells (RBC) or hemoglobin in circulation.1 Chronic kidney disease (CKD), cancer, inflammatory disorders, nutritional deficiencies, genetic disorders, and some drugs can cause anemia. Nutritional deficiency anemia is frequent in women, children, and elderly. Iron and vitamin supplementation is used to treat nutritional deficiency anemia.2 Thalassemia is a most common genetic disorder causing anemia.3 Chronic kidney disease, chemotherapy, and inflammatory diseases can reduce erythropoietin (EPO) synthesis and inefficient iron homeostasis.1,4 Treatment of anemia associated with CKD is done using EPO analogues or erythropoiesis-stimulating agents (ESA).5,6 However, they are associated with cardiovascular side effects and may increase the progression of malignancy in cancer patients.7 CKD patients also demonstrate iron deficiency which requires iron supplementation. 8 Functional iron deficiency is a significant problem that limits the use of ESAs in most of the patients.9 Stabilization of hypoxia-inducible factor (HIF) represents a novel approach for the treatment of CKD. Under normal conditions, HIF undergoes oxidative degradation by prolyl hydroxylase domain (PHD) enzymes. Hypoxia is the major inhibitory factor for PHD activity, which stabilizes HIF and stimulates EPO synthesis. PHD inhibition also decreases hepcidin production, which improves iron utilization.10 An increase in EPO and a decrease in hepcidin, caused by pharmacological inhibition of PHDs, can efficiently cure anemia associated with chronic diseases.

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MECHANISM OF ERYTHROPOIESIS Erythropoiesis occurs in the bone marrow. In the bone marrow, hematopoietic stem cells (HSCs) differentiate into colony forming units-erythroid (CFU-Es) and then to erythroblasts, © 2018 American Chemical Society

Received: November 15, 2017 Published: May 1, 2018 6964

DOI: 10.1021/acs.jmedchem.7b01686 J. Med. Chem. 2018, 61, 6964−6982

Journal of Medicinal Chemistry

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Figure 1. Stages of erythropoiesis. The process of erythropoiesis occurs in the bone marrow. Erythropoiesis is the process of proliferation and differentiation of erythroid progenitors into reticulocytes and erythrocytes (RBC). The common myeloid progenitors, also known as stem cells, form RBC, white blood cells, and platelets. Stem cells are differentiated into early stage burst-forming unit−erythroid (BFU-E) and then later stage colony-forming unit−erythroid (CFU-E) progenitors. CFU-Es differentiate into proerythroblasts, which then undergo mitosis to form erythroblasts. The erythroblast is basophilic, polychromatic, and orthochromatic. The erythroblast denucleates to become a reticulocyte, which enters the bloodstream. Reticulocytes mature to become RBCs after shedding cellular organelles such as mitochondria, Golgi apparatus, and endoplasmic reticulum. A stage of erythropoiesis from BFU-E to erythroblast differentiation requires EPO. Maturation of RBCs from erythroblasts requires iron.

Figure 2. PHD-HIF mechanism of enzyme action. At normal oxygen concentration (normoxia), prolyl hydroxylase domain-containing (PHD) enzymes (PHD1, PHD2, or PHD3) are active. Active PHDs hydroxylate HIF-α residues. Once hydroxylated, HIF-α is recognized by Hippel−Lindau tumor suppressor protein (pVHL), which facilitates its proteasomal degradation by E3 ubiquitin ligase. Thus, E3 ubiquitin ligase does not cause degradation of HIF but rather ligates HIF, thereby tagging it with a signal to carry it to proteasome for degradation. For this reaction, PHDs require 2-oxoglutarate (2-OG) as a substrate and iron (Fe) as a cofactor. On the other hand, in hypoxic conditions, or by pharmacological inhibition of PHDs, HIF-α is protected from degradation and heterodimerizes with HIF-β. The HIF complex is then translocated to the nucleus and stimulates EPO gene expression. Stimulation of EPO gene expression under hypoxia or PHD inhibition translates into efficient erythropoiesis. HIF complex is also involved in the regulation of angiogenesis, glucose metabolism, lipid metabolism, and vasodilation.

HIF-2α) by prolyl hydroxylase (PHD). Once hydroxylated, HIF-α is recognized by Hippel−Lindau tumor suppressor protein (pVHL), which facilitates its proteasomal degradation by E3 ubiquitin ligase. Thus, E3 ubiquitin ligase does not cause degradation of HIF but rather ligates HIF, thereby tagging it with a signal to carry it to proteasomes for degradation. PHD enzymes require oxygen, iron, ascorbate, and 2-oxoglutarate (2OG) for their activity (Figure 2). Oxygen deficiency (hypoxia) causes PHD inhibition and activates HIF-α, while HIF-β is constitutively expressed.18 There are three isoforms of PHD, namely, PHD1, PHD2, and PHD3.18 The hypoxic stabilization of PHDs also follows a negative regulatory mechanism. In prolonged hypoxia, PHD3, and frequently PHD2 mRNA, is increased to accelerate degradation of HIF upon reoxygenation after long-term hypoxia.18 The active sites of all isoforms of PHDs share a high-sequence homology. PHD1 shows more affinity toward

morphogenetic protein/SMAD (BMP/SMAD) signaling. On the other hand, increased inflammation stimulates hepcidin production via IL-6/STAT signaling.8,16

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STIMULATION OF ERYTHROPOIESIS BY HIF-PROLYL HYDROXYLASES High altitude or hypoxia enhances RBC synthesis by stimulating EPO production. Hypoxia-inducible factor (HIF) is the regulator for hypoxic induction of erythropoiesis.17 There are three isoforms of HIF (HIF-1, -2, and -3), of which HIF-1 and HIF-2 regulate the vascular response to hypoxia. Specifically HIF-1 regulates the metabolic response, while HIF-2 stimulates erythropoiesis in response to hypoxia. HIF-3 is less closely related to HIF-1 and HIF-2, and its role is not yet fully understood. HIF-1 and HIF-2 consist of two subunits, namely α and β. HIF-α is hydroxylated at two proline residues (Pro402 and Pro564 for HIF-1α, and Pro405 and Pro531 for 6965



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Figure 3. Iron metabolism and the role of hepcidin. Absorption of iron mainly occurs in the intestine. The nonheme iron in the ferric form is converted in the ferrous form by duodenal cytochrome b (Dcytb). Divalent metal transporter 1 (DMT1) facilitates the transfer of iron into enterocytes. Hepcidin is a regulator of iron homeostasis that downregulates DMT1 and limits iron absorption. Ferritin is the soluble and nontoxic form of iron in plasma. Ferritin is the form of stored iron. Iron in the ferrous state enters into blood circulation with the help of the transport protein ferroportin. Hephastin converts ferrous iron to ferric iron, which then binds to transferrin (TRF) and circulates as TRF bound ferric iron. It is taken up into the metabolic tissues such as macrophage or the liver by transferrin receptor (TRFR). TRF-bound ferric iron is then released free into the cell. Inside the cell, DMT1 converts ferric iron into ferrous iron, after which it is stored as ferritin or released from the cell by ferroportin. Hepcidin inhibits ferroportin, by which it can block iron uptake into cellular stores and decreases iron in circulation. Like hephastin, ceruloplasmin present in blood converts ferrous iron into the ferric form. The ferric form binds to TRF and circulates in the blood. TRF-bound ferric iron is taken up by reticulocytes for hemoglobin synthesis, which then mature into RBCs.

DMT1. Ceruloplasmin is the enzyme in the enterocyte that oxidizes ferrous iron back to the ferric form.16 Transferrin transports iron (in the ferric form) in plasma using the transferrin receptor.16 Heme-oxygenase-1 required for the release of iron from the reticuloendothelial system and ferroportin is the membrane iron exporter present in all major iron depots in the body.17 Although HIF has a direct role in modulation of all of these proteins involved in iron metabolism, the primary mechanism by which it indirectly stimulates efficient iron utilization is through regulation of hepcidin. Stabilization of HIF-α by oxygen deficiency or genetic changes or pharmacological inhibition leads to suppression of hepcidin.8 Apart from liver and kidney, the PHD-HIF system has an important role in the bone marrow. HIF activation stimulates expression of the EPO receptor and also modulates hemoglobin synthesis. Also, it modulates the differentiation and maturation of erythroid cells.27 Peritubular interstitial cells generate EPO in the kidneys.28 With increasing hypoxia, the density of the EPO producing cells increases substantially, as a result of the increased density of EPO producing cells, which results in increased EPO gene expression and enhanced release of EPO into the bloodstream.29 Differentiation of renal fibroblast-like cells under inflammatory stimulus results in fibrosis and, hence, decreased ability of the cells to generate and release EPO into plasma,

HIF-2α than HIF-1α and hydroxylates it under normoxic condition. Deletion of the PHD1 gene induces hypoxia tolerance by shifting the tricarboxylic acid cycle to the glycolytic pathway.19 Tolerance to hypoxia is due to reduced oxidative stress and may not be due to angiogenesis, erythropoiesis, or vasodilation.20 PHD2 gene deletion accelerated RBC synthesis without a significant increase in EPO levels.21 Inactivation of PHD1 and PHD3 leads to erythrocytosis by activating the hepatic HIF-2α, whereas only PHD2 deficiency leads to erythrocytosis by activating the renal EPO pathway.22 Interstitial fibroblasts of kidneys express PHD enzymes. PHD2 inhibition causes HIF-2α stabilization and generates EPO.23 Fibrosis decreases the capacity of the renal tissue to generate EPO, sequentially causing anemia.24 HIF stabilization can also cause EPO synthesis in the liver. In the kidneys and liver, HIF-2 stimulates EPO gene transcription by binding to hypoxia-responsive regulatory elements.25 Erythropoiesis requires EPO as well as iron for successful maturation of RBCs. HIF-2 induces efficient absorption as well as utilization of iron by stimulation of genes required for iron transport and metabolism (Figure 3). HIF-2 stimulates divalent metal transporter 1 (DMT1) and duodenal cytochrome b reductase 1 (Dcytb).26 Dcytb converts the ferric iron to the ferrous form (Fe2+) in the intestine. This conversion is vital because the gut lumen cannot absorb the ferric form of iron. Once the iron is converted to the ferrous form, it is taken up into enterocytes by 6966



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which results in anemia.30 In the liver, GATA-4 is the major modulator for EPO production.30 It has been observed that the induction of erythropoiesis in the liver causes normalization of hemoglobin in rodents, in which anemia was induced by nephrectomy or inflammation.31 It has been observed that inactivation of all the three PHD isoforms is needed for a significant EPO synthesis from liver.32 CKD is associated with pathologies like enhanced levels of creatinine and urea in the circulation. These pathologies can modulate the HIF system, ultimately adding to the burden of anemia.33 Positive changes in renal physiology can also add to renal EPO production, as observed with diuretic treatment.34 A major population of CKD patients suffers from severe anemia. The degree of anemia in these patients is dependent on the severity of renal failure.35 However, even the fibrotic kidneys have the potential to generate EPO under a positive stimulus, which can correct anemia.35 A substantial population of CKD patients does not respond to ESA therapy due to an underlying inflammatory state. Inflammation-induced functional iron deficiency is a common reason for the unresponsiveness or resistance of CKD patients to EPO therapy.36,37 The release of inflammatory cytokines decreases not only renal EPO production but also suppresses efficient differentiation and maturation of erythropoietic progenitors in the bone marrow.37 Inflammation upregulates hepcidin through the STAT-3 pathway or the BMP/SMAD1/5/8 pathway.8 As already discussed, increased hepcidin causes degradation of ferroportin and causes entrapment of iron in tissue stores, resulting in a functional iron deficiency, which cannot be corrected using oral iron supplementation. As the iron deficiency gets severe, iron regulatory proteins bind to HIF2α and further decrease erythropoiesis.38 This feedback mechanism makes the anemia in CKD patients difficult to treat using erythropoietin analogues or iron supplementations. Activation of HIF signaling using PHD inhibition demonstrates a unique and rational approach to correct anemia associated with chronic diseases. Because of the pharmacological inhibition of HIF-PHD, there occurs a transient induction of HIF-regulated genes. Plasma EPO generated due to this inhibition is much lower than that observed in patients after injection of recombinant erythropioetins,39 suggesting that PHD inhibitors may not have the risk for cardiovascular side effects that occur due to high levels of plasma EPO. Prolyl hydroxylase inhibitors decrease hepcidin and thus reduce the functional iron deficiency in CKD patients.39,40 Currently, six PHD inhibitors are in various phases of clinical trials for the treatment of anemia associated with CKD.41 FG2216, the first clinically tested small molecule PHD inhibitor, has shown enhanced erythropoiesis in patients with end-stage renal disease (ESRD).42

compounds are not specific PHD inhibitors. They also inhibit iron-dependent pathways other than PHDs and may cause undue toxicities. Synthesis of 2-OG (1) analogues was the first specific approach used for the design of PHD inhibitors. Most of the clinically advanced molecules are 2-OG derivatives. PHD enzymatic activity requires 2-OG as the substrate. A cocrystal structure of 2-OG bound to the PHD2 catalytic domain (PDB 3OUJ) has shown bidentate chelation of the Fe center by the C1 and C2 sp2 oxygen atoms and a salt bridge between the C5 carboxylate and Arg383. The wall of the 2-OG pocket is lined with hydrophobic residues including tyrosine-310, -303, and -329, isoleucine-327 and -256, methionine-299, leucine-343, tryptophan-389, alanine-301 and -365, and valine-376. NOxalylglycine (2, NOG) was the first reported 2-OG mimetic molecule.48 Compound 2 shows a stronger interaction with PHD2 (PDB 3HQR) and has an N−H group in place of the C3 methylene group in 1. This modification reduced the susceptibility of the 2-carbonyl group to nucleophilic attack by oxygen. Dimethyloxalylglycine (3, DMOG)49 is a cellpermeable precursor of NOG used extensively as a tool compound to study the in vivo pharmacology of PHD inhibitors. Several active site targeted inhibitors identified in this class have strong binding interactions with hydrophobic residues in the 2-OG pocket.50 Hydrophobic heterocycles used in the place of ferrous ion coordination fragment of 2 caused a stronger binding with PHD. A crystal structure (PDB 2HBT, Figure 4) of isoquinoline derivative 15 (R1 = Cl, R2 = R3 = R4 = R5 = H in 4, Figure 5)

Figure 4. Structure of a 247-residue C-terminal catalytic domain of PHD2 bound to 15 (PDB 2HBT).

bound with PHD was reported by a group at Procter and Gamble.51 Groups at Oxford University and Amgen have disclosed the crystal structure (PDB 2G1M) of another isoquinoline derivative (R1 = R2 = R3 = R4 = H, R5 = I in 4, Figure 5).52 Three conserved iron-binding triad residues (His313, Asp315, His374) occupy half of the ligand sphere of the bound Fe (Figure 4). The other three metal coordination sites of the octahedral complex involve the bidentate binding of the isoquinoline nitrogen atom and the exocyclic carbonyl oxygen atom of the ligand and a water molecule. The terminal carboxylate group interacts with Arg383 by making a salt bridge and with Tyr329 in the back of the 2-OG binding pocket by a H bond interaction. A π-stacking hydrophobic interaction with Tyr310 and a H-bonding interaction of the phenolic hydroxyl group with Tyr303 are other key interactions observed for this molecule.

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EVOLUTION OF MEDICINAL CHEMISTRY STRATEGIES FOR PHD INHIBITION Under normoxic conditions, PHDs catalyze the hydroxylation of two conserved proline residues in HIF-α. The hydroxylation of HIF-α directs it toward degradation. Because metal ions are required for the 2-OG-dependent hydroxylation, targeting or chelating metal ions was the first approach exercised for the design of PHD inhibitors. Salts of Co2+, Cu2+, and Ni2+ were used to antagonize Fe2+, a cofactor for PHD enzyme action.43 Additionally, iron chelators such as deferoxamine,44 3,4dihydroxybenzoic acid,45 1,10-phenanthrolines,46 and quercetin47 have been shown to inhibit PHD. However, these 6967



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Figure 5. Chemotypes derived from 2-OG.

An isoquinoline scaffold (4, Figure 5)56 is widely used in the design of PHD inhibitors, especially in two clinical candidates, 15 (FG-2216, Figure 6) and 16 (FG-4592, roxadustat, Figure 6), discovered by FibroGen, Inc. (San Francisco, CA, U.S.). Several research groups modified the phenyl ring of the isoquinoline core with 5,6-membered bicyclic, tricyclic, and heterocyclic rings.50 Monocyclic pyridine derivatives (represented by general structure 5) were developed by Akebia Therapeutics.57 The oxygen atom from carbonyl group of the quinolone (e.g., 6) and monocyclic pyridone (e.g., 7) derivatives was useful for Fe chelation.50 The phenyl ring of the quinolone core underwent modification in these compounds for optimizing the activity.50 The compounds discussed above contain a glycinamide side chain attached to the ring containing the heteroatom that chelates iron. Modifications where the iron chelating heteroatom is present in another ring fused with a central core were also reported. GSK has reported some such compounds like quinolines (X = CH in 8),58 quinoxalines (X = N in 8)59 with 6-membered ring fusion, and benzimidazole derivatives (e.g., 9 and 10)60,61 with a 5-membered ring fusion. In compounds denoted by 10, an additional cyclic ring fused with the central core has replaced the phenolic OH. It has the potential to produce symmetrical analogues which offer two chelation binding modes. Janssen Pharmaceuticals reported

The newer 2-OG analogues caused a specific PHD inhibition owing to a differentiated mechanism. 2-OG mimetics like 2 allowed HIF binding with PHD enzymes but blocked the formation of the Fe(IV)O complex (PDB 3HQR). On the other hand, larger heterocyclic inhibitors such as 15 stabilized a closed conformational structure, preventing HIF substrate binding (PDB 3HQU).53 Most of the PHD inhibitors consists of three structural features based on the ligand−protein interactions. The first characteristic is a bidentate coordination site to an iron atom. The second important feature is a carboxylic acid forming a salt bridge with the Arg383 side chain. The third attribute is a hydrogen bond acceptor for the phenolic hydroxyl of Tyr303. Figure 5 demonstrates these features. However, several potent PHD inhibitors identified through high throughput screening (HTS) do not possess a carboxylic acid group that can form a salt bridge with Arg residue.50 Chan et al. described their efforts to identify PHD inhibitors that do not contain the carboxylic acid group.54 Stroke was the primary indication of these PHD inhibitors. The lack of a carboxylic acid group helps in better CNS penetration, and hence these inhibitors were considered useful in the treatment of neurological disorders. A comprehensive review has compiled the assay procedures used to screen PHD inhibitors.55 A separate review has covered significant 2-OG derived PHD inhibitors.50 6968



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Figure 6. Carboxylic acid-based PHD inhibitors (part 1).

benzimidazole derivatives represented by 11.62 An X-ray cocrystal structure with one of the compounds (R1 = R4 = H, R2 = R3 = Cl) with PHD2 (PDB 3OUI) has shown that the NH group from the imidazole ring interacts with Tyr303. In earlier compounds, this purpose was served by the key phenolic OH. The other nitrogen of the imidazole ring is involved in Fe chelation with carbonyl group from the glycinamide side chain. Cyclic modification of the amide group in pyrazole derivatives caused strong iron chelation (12). JNJ-42041935 (R1 = R4 = H, R2 = OCF3, R3 = Cl in 12, Figure 5) is a pan-PHD inhibitor whose pharmacological profile has been reported.63 Expansion of the benzimidazole core in 12 by insertion of the “CO” group into the C−N bond allowed a H-bond interaction with Tyr303 as in a series of quinazolinones (13).64 JNJ-42905343 (R1 = R4 = H, R2 = 2,6-dimethylphenoxy, R3 = F in 13) increased serum iron and erythropoietin in rats.65 In PGPS (peptidoglycan-polysaccharide)-treated rats, administration of JNJ-42905343 for 28 days corrected functional iron deficiency (FID) and anemia, an effect attributed to the increased

expression of iron reductase Dcytb and iron transporter protein DMT1 in the duodenum. Human recombinant EPO (rhEPO) did not affect Dcytb and DMT1 and was not effective in correcting anemia in the PGPS model.65 The aminoquinoxaline derivatives 14 demonstrate a significant modification in which a substituted amino group replaced the carbonyl group that forms a H bond with Tyr303.66 As discussed above, inhibitors derived from 2-OG possess a carboxylic acid group as a critical pharmacophore, while noncarboxylic acid-based PHD inhibitors are also reported (mainly identified through HTS). In this review, we have classified them as carboxylic acid-based PHD inhibitors, noncarboxylic acid PHD inhibitors, and even in the third category where they have demonstrated a nonconventional binding mode especially with iron.

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CARBOXYLIC ACID-BASED PHD INHIBITORS The first optimized structural modification of 2-OG derived chemotypes resulted in 15 (FG-2216, Figure 6). FibroGen advanced this molecule to phase II trials in 2005.67 Compound 6969



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Figure 7. Carboxylic acid-based PHD inhibitors (part 2).

15 caused enhanced erythropoiesis in preclinical studies as well as in patients. The erythropoiesis was higher in nephric dialysis patients than in anephric patients, implying that 15 induced EPO production in malfunctioning kidneys. However, following the death of one subject due to fulminant hepatitis in the trial, clinical development of 15 was suspended.68 Another clinical candidate is 16 (roxadustat, FG-4592), an analogue of FG-2216 with an additional 7-phenoxy substituent and methyl group in place of the chloro in 15.69 Compound 16 is currently in phase III clinical trials. Zhejiang Beta Pharma Inc. also reported the polymorphic forms of 16.70 Fibrogen reported PHD1 selective isoquinoline derivatives for therapeutic indications such as muscle degeneration, colitis, and inflammatory bowel disease (IBD) exemplified by 17− 20.71 This patent application describes data for 184 compounds that show selective inhibition of PHD1 over PHD2. The prototype compound 17 was nonselective, and selectivity was enhanced by chain elongation, e.g., 18, which was 5-fold selective for PHD1, although further elongation did not improve the selectivity. Branching the alkyl chain by

introducing a methyl group in 19 made it 40-fold selective for PHD1 over PHD2. New branching with alkyl, aryl, and aralkyl groups at both the amino and carboxylic acid terminals did not improve selectivity. Compound 20, with dimethyl branching and methoxy substituent on phenyl ring, has shown excellent activity and 40-fold selectivity (half-maximal inhibitory concentration IC50 PHD1 = 0.24 μM, PHD2 = 9.4 μM). Shenyang Sunshine Pharmaceuticals in two separate publications reported 5-hydroxy-1,7 naphthyridine derivatives exemplified by 21 and 22. Compounds 21 and 22 are aza analogues of isoquinoline derivatives of fibrogen (e.g., 16) substituted with aryl/heteroaryl and aryloxy/heteroaryloxy groups, respectively.72,73 These compounds were evaluated for EPO induction and PHD2 inhibitory activity in preclinical assays and compared with 16, where compounds 21 and 22 have shown superior activity in of reticulocyte production. Procter and Gamble invented monocyclic pyridines exemplified by 23 and 24 and later licensed to Akebia therapeutics.74 Compound 23 (also known as vadadustat) is now undergoing late phase clinical trials for the treatment of 6970



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13.4, PHD3:5.5 ± 5.1) with at least 1000-fold selectivity against collagen prolyl hydroxylase (CP4H) and factor inhibiting HIF (FIH) in enzyme inhibition assays. When dosed intravenously, 34 showed low blood clearance in the mouse, rat, dog, and monkey and good oral bioavailability across all of these species. Compound 34 was dosed at 60 mg/kg in B6D2F1 mice to see its effect on EPO. Following treatment for a week with 34, EPO protein levels peaked at 12 h postdose by 11.2-fold with negligible change in VEGF. Compound 34 was able to increase reticulocytes and hemoglobin in B6D2F1 mice in a dose-related manner within a week’s treatment. Compound 34 was found to be safe in 14-day oral toxicity studies in Sprague−Dawley rats. Taisho Pharmaceuticals report a series of partially saturated pyridone derivatives exemplified by 35.91 This compilation reports the IC50 values of selected compounds and the percentage inhibition of PHD2 at 1 μM for nearly 500 compounds. Lead optimization involved various alkyl, aryl, and heteroaryl substituents at the nitrogen atom of the pyridone ring. Alkyl groups are connected to both saturated carbons of the central core. Spirocyclic modification of the central core was done using 3−6-membered carbocyclic or heterocyclic ring systems. Compound 35 show the most potent inhibition of PHD2 with an IC50 of 14 nM. N-Alkoxyquinolones exemplified by 36 were disclosed by Cadila Healthcare.41,92 Compounds in this invention increased EPO and hemoglobin levels after oral administration to mice. Pyridine, pyrimidine, and thiophene groups were used to replace the phenyl ring. An optimized molecule 36 (ZYAN1, desidustat) is now in phase 2 clinical trials.92,93 Merck reported a series of bicyclic pyridones exemplified by 37−40.94 The fused ring was either a fully saturated cyclohexyl ring or a saturated ring with additional N and O heteroatoms. Eighty-five compounds in this report are potent PHD2 inhibitors. Structure−activity relationships (SAR) were evaluated by exploring various aryl, aralkyl, or heteroaralkyl groups at the N atom of the pyridone ring. Tetrahydropyran derivative 37 is the most potent PHD2 inhibitor (IC50 = 5.4 nM) in this group. Shifting the oxygen atom to its adjacent carbon did not change the potency for PHD2 inhibition. Another modification with fused cyclohexyl ring as in 38 inhibited PHD2 with an IC50 of 66 nM. Introduction of NH as in 39 has improved the potency for inhibition of PHD2 (IC50 = 22.9 nM). The lactam derivative 40 demonstrated improved potency (IC50 = 7.3 nM). Varying the substitution pattern of groups attached to the N atom of pyridone ring was responsible for the change in PHD2 inhibitory activity. Merck has reported a set of compounds in the quinolone class exemplified by 41−46.95 Piperidine, morpholine, thiomorpholine, and piperazine rings were fused to the quinolone to generate a tricyclic central core. The phenyl ring of the quinolone was substituted with alkyl, aryl, halogen, −CN and − SO2CH3 groups, with maximum activity observed with polar groups −CN and − SO2CH3 at almost all positions in the majority of compounds. Aryl, aralkyl, and heteroalkyl groups are connected to both carbons. Morpholine-fused tricyclic derivative 41 inhibited PHD2 with an IC50 of 21 nM. The introduction of a CN group, as shown in 42, improved PHD2 IC 50 to 1.8 nM. Compound 43 with a 4trifluoromethylphenyl group inhibited PHD2 with IC50 values of 0.9 and 1.3 nM for the separated enantiomers. Its corresponding thiomorpholine 44, piperazine 45, and piperidine 46 derivatives inhibited PHD2 with IC50 values 1.5, 1, and 1.9 nM, respectively, in racemic form.

anemia associated with CKD. A fluoro analogue 24 (AKB6899) has the potential to treat cancer.75 It was shown to be a stabilizer of HIF-2α via inhibition of PHD3, which enhances the production of suppressor of vascular endothelial growth factor receptor-1 (sVEGFR-1) but not vascular endothelial growth factor (VEGF) from granulocyte-macrophage-colonystimulating factor (GM-CSF)-stimulated monocytes and macrophages. Compound 24 suppresses angiogenesis and provides a potential method for treating cancer. Compound 24 caused selective up-regulation of HIF-2α but did not affect VEGF. An increase in HIF-2α also enhanced sVEGFR-1 production in human peripheral blood monocytes when stimulated with GM-CSF in the presence of 24. Recent reports indicate the polymorphic forms of 23 and a process for the preparation of 23, 24, and analogues.76,77 Recently, both of these compounds were shown to be useful for the treatment of ocular diseases,78 followed by two disclosures studying formulation and dosing regimens for both the compounds79 and the synthesis of deuterium-enriched analogues.80 In the same monocyclic pyridine class, China Pharmaceutical University reported alkynylpyridines as PHD inhibitors (e.g., 25).81 Compounds in this series possess a substituted alkynyl group on the pyridine ring of 23 and 24. Compound 25 inhibited PHD2 with an IC50 of 13.7 nM. Another modification of the pyridine class was reported by Shenyang Sunshine Pharmaceuticals in the form of diaryl ether analogues of compounds 23 and 24, in which a phenoxy group replaced the aryl substituent (e.g., 26, PHD2 IC50 = 15 μM). Mitsubishi Tanabe reported pyrazolopyrimidine derivatives exemplified by 27−29.82 These compounds are the heterocyclic modification of phenyl ring of 13 (Figure 5) described earlier by Janssen. In this disclosure, PHD2 and PHD3 enzymatic inhibition data of more than 500 compounds are reported, along with EPO production measured in a cell-based assay. The structure−activity modifications involved the use of branched alkyl, cycloalkyl, and aralkyl groups on the pyrazole ring. Compound 27, with a bulky bis(4-chlorophenyl) methane group, inhibited PHD2 with an IC50 value of 0.02 μM and PHD3 with 2.6 μM, demonstrating 130-fold selectivity for PHD2. The inhibition of PHDs correlated with a 40-fold increase in EPO, whereas compound 28 with a naphthyl substituent (PHD2 IC50 = 0.012 μM, PHD3 IC50 = 0.18 μM) has been shown to increase EPO induction by more than 100fold. Compounds with substituents on the other nitrogen, as described in 29, have usually been found to have a negligible effect on EPO induction despite higher potency for PHD inhibition. Merck has reported bis-carboxamide derivatives exemplified by 30, 31, 32, and 33.83,84 Compound 30 was a potent PHD2 inhibitor (IC50 < 20 nM). In a separate publication, biscarboxamide derivatives with a pyridine central core were reported (e.g., 31, IC50 = 0.62 nM).85,86 Compound 32 was designed to increase the half-life of the pyrimidine class of compound by inserting a cyclopropyl group (e.g., 30, half-life of 0.28 h, and 32, half-life of 4.78 h).87,88 A prodrug approach is also useful for the improvement in oral bioavailability.89 An ester prodrug 33 was found to have 3-fold increased bioavailability compared to that of the parent acid 32 in Wistar rats when administered orally at 2 mg/kg dose. GSK has reported the pyrimidine-trione class of inhibitors.50 A clinical candidate, 34 (daprodustat, Figure 7), from this class is in late phase clinical evaluation.90 It is a potent inhibitor of all PHD isoforms (IC50s (nM) for PHD1:3.5 ± 0.6, PHD2:22.2 ± 6971



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Figure 8. Non-carboxylic acid PHD inhibitors (part 1).

a H-bonding interaction with the OH with Tyr303. Compound 54, with a 2-phenylethyl side chain, displayed much better activity (IC50 PHD2 = 0.22 μM, EC50 Hep3B = 5.7 μM) and exhibited a significant increase in EPO levels in mice (plasma EPO = 6500 pg/mL) and rat (plasma EPO = 17000 pg/mL) at 8 h when administered orally at a dose of 10 mg/kg. This compound had good solubility and did not affect CYP enzymes. In rats, it was shown to have a short t1/2 and claimed to exhibit good oral bioavailability (F(%) = 75). Oral dosing of compound 54 at 1 and 3 mg/kg showed a dose-dependent increase in hemoglobin following a month-long treatment in rats. Compound 55, with a methyl substituent in the 5membered ring, showed a reduction in activity compared to 54, suggesting possible interference with the adjacent N atom involved in the iron chelation (IC50 PHD2 = 1.6 μM, EC50 Hep3B = 15 μM).

Merck developed dihydrothienopyridone and dihydrofuropyridone series of PHD inhibitors, exemplified by 47 and 48.96 Compounds in these series contain aryl or heterocyclic groups directly attached to N atom of pyridone ring unlike previously reported compounds where the alkyl group is attached to it. Compound 47, with an IC50 value of 5.8 nM, and compound 48, with an IC50 value of 4.1 nM, are the optimized compounds. Additional work in a dihydrofuropyridone class of inhibitors was reported focused on modulating the pharmacokinetic (PK) profile. This report disclosed PHD2 inhibitory activity and PK data of 60 compounds in preclinical models.97 Compound 49 and 50 have been shown to possess significantly higher hepatic availability with liver/plasma ratio of drug concentration of 35.65 and 257.5, respectively. In a recent publication, a group at Japan Tobacco disclosed a series of triazolopyridine derivatives (e.g., 51−55) and the subsequent discovery of their clinical candidate 54 (JTZ951).98 Although 51 inhibited PHD (IC50 = 0.82 μM), it failed to stimulate EPO release from Hep3B cells. It was claimed to be due to poor cell permeability of this compound. An improvement in cell permeability was achieved by incorporation of lipophilic substituents on the phenyl ring of the central core. The optimized compound was 52, which has an npentyl substituent (IC50 PHD2 = 0.85 μM, EC50 Hep3B = 4.2 μM). Inhibitory potency was reduced when the n-pentyl group was attached next to the phenolic carbon (e.g., 53, IC50 PHD2 = 2.5 μM, EC50 Hep3B = 9.3 μM). suggesting a possible loss of

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NON-CARBOXYLIC ACID PHD INHIBITORS HTS efforts by GSK led to the discovery of quinazoline-2,4diones and 4-oxo-2-thioxo-7-quinazolines exemplified by 56, 57, 58, and 59 (Figure 8). All compounds in these series had either a pyrimidinedione or ketopyrimidinethione core attached to an N3-azaheterocycle.99,100 These compounds bind to the active site iron by chelation of the N atom of the heterocycle and either the keto or thiono group of the core. The phenyl ring was modified to a pyridine ring as in 57101 and a thiophene ring as in 58 and 59.102,103 PHD3 inhibitory activity (IC50 6972



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Figure 9. Noncarboxylic acid PHD inhibitors (part 2).

of cytochromes P450. It was found to be selective against an array of enzymes and receptors and possessed good oral bioavailability and a longer half-life in all of the preclinical models, including rhesus monkeys. Despite the increase in EPO levels, VEGF did not change after treatment with 63 in mice. In Sprague−Dawley rats, this compound caused an increase in EPO and hemoglobin after repeated dosing, at 1.5, 5, and 15 mg/kg, by the oral route. The 5-aminocarbonyl-4-hydroxypyrimidine class contained compounds bearing phosphinic acid, phosphonic acid, and phosphonate ester groups, exemplified by 64.109,110 Compound 64 inhibited PHD2 with an IC50 in the low nanomolar range. Phosphorus-containing groups are attached to one of the phenyl rings of the benzhydryl moiety, while a few derivatives also replaced one of the phenyl rings with aliphatic phosphonic acid derivatives. A novel spiroindolone series exemplified by 65, with one more report of spiroazaindoles, was reported by Merck.111,112 A major effort using mass spectrometry-based high throughput screening methodology assessed more than 500000 compounds.113 Optimization of early hits led to the identification of compound 65 (PHD2 IC50 = 0.7 nM), in which a 2pyridylmethyl group was found to be essential for the desired potency, although it was not bioavailable. Efforts to reduce the lipophilicity of the central core resulted in a spirohydantoin class of compounds, exemplified by 66. It showed high potency (IC50 PHD1 = 0.2 nM, PHD2 = 0.2 nM, PHD3 = 1.6 nM), improved PK (F(%) = 47%, t1/2 = 1.1 h in rat), and minimal hERG inhibition (IC50 = 22 μM).114 Compound 66 is a short-acting PHD inhibitor, which was based on the rationale that only a very short-time inhibition of the PHDs is sufficient for HIF stabilization and further downstream EPO upregulation. A shorter duration of action offered by the shorter half-life of the compound may lead to an improved safety profile in the clinic. The concept of having a short half-life of PHD inhibitors was also discussed by researchers at Janssen by defining duodenal uptake of iron as

value) for all exemplified compounds was in the range of 1− 100 nM. Akebia has reported hydroxypyridone derivatives 60 and 61 derived from a known iron-chelator L-mimosine.104 Compound 60 has been reported to inhibit PHD2 with an IC50 of 14 μM. These compounds were claimed to promote wound healing, increase innate defense, and treat ulcerative colitis.105 Merck reported 5-aminocarbonyl-4-hydroxypyrimidine derivatives 62 and 64 identified by HTS.106,107 Most of the reported compounds are potent (IC50 of 60 h) in dogs and monkeys. To achieve a once daily dosing paradigm in humans, strategies to increase phase I metabolism and hence lower the residence time was adopted. The introduction of two methoxy groups resulted in compound 63 with a significantly lowered t1/2 observed in the dog (19 h) and monkey (10 h). The reduced half-life of 63 was due to O-demethylation. A pyridazine ring provided an iron chelation site and was found useful to remove human ether-a-go-go-related gene (hERG) inhibitory activity. A pyrazole ring caused hERG activity inhibition (hERG inhibition IC50 = 0.9 μM). Compound 63 inhibited PHD2 with an IC50 of 1 nM and was found not to be a substrate, inhibitor, or inducer 6973



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a target to treat FID and inflammation-induced anemia.65 Oral administration of a low dose of a PHD inhibitor is an effective treatment for FID without the requirement for systemic exposure to the compound. The local concentration of the PHD inhibitor that surrounds the enterocyte of the duodenum is higher after oral administration than that achieved after systemic absorption. In this context, a PHD inhibitor that has limited systemic absorption and one with a short half-life might be advantageous for the treatment of FID and anemia associated with chronic disease.65 A spirocyclic class of PHD inhibitor was also explored by GSK, which resulted in the identification of 2,8-diazaspiro[4.5]decan-1-one derivatives, exemplified by 67 (pIC50 = 8.3).115 This series of compounds lacks the phenyl ring fusion as seen in 65. A crystal structure between a close analogue of 67 and PHD2 revealed several novel interactions, including a H-bond between the lactam carbonyl and Arg322, a π−cation interaction with Arg322, a π−π stacking interaction with Trp389, and a π−π stacking interaction with His313. In an oral absorption study in mice, 67 demonstrated good oral bioavailability with high peripheral exposure (dose-normalized area under curve (DNAUC): 634 ng h/mL). The high oral bioavailability of 67 was due to low hepatic clearance. Compounds 68−70 possessing a pyrazolone central core (Figure 9) were earlier disclosed by Bayer.116,117 Compounds from this series chelate iron through the nitrogen from the pyridine or pyrimidine ring attached via the nitrogen atom of the central core and N1 of the pyrazolone ring in its enol tautomeric form. The enolic OH may engage Tyr303 as observed with phenolic PHD inhibitors. The nitrogen of the 3pyridyl ring in 68−69 was essential for high potency. Compound 68 inhibited PHD activity in a FRET assay with an IC50 value of 0.43 μM and the EC50 value in a luciferase reporter assay in A549 cells was found to be 4.9 μM.116 The IC50 value of amide analogue 69 was found to be 0.91 μM. Bayer has also described molecules similar to 68−69 with a dihydropyrazolethione (not shown) as a central core with similar activity.118,119 Bayer has disclosed a pyrazolone series replacing the 3-pyridyl with 5-membered nitrogen-containing heterocycles, as exemplified by 70.117 A recent publication disclosed the discovery efforts for the clinical candidate 70 (molidustat, BAY 85-3934).120 A brief SAR was drawn around the triazole ring, pyrazole central core, and pyrimidine ring. Replacement of the triazole ring with other heterocycles such as imidazole, thiadiazole, and isooxazole with an N atom always present at the third position suggested the importance of a heteroatom for optimum potency. Docking of the pyrazolol tautomer of 70 into the active site of PHD2 (PDB 2G19) suggested bidentate iron binding via the ring nitrogen atoms of the pyrazolol core and the adjacent pyrimidine ring. The triazole moiety was proposed to engage in an electrostatic interaction with Arg 383. It was shown to have excellent oral bioavailability across the species (F (%): 34 (rat), 71 (dog), and 61 (monkey)). The metabolism of 70 was found to occur via N-glucuronidation. Urinary excretion is the primary route of elimination of 70.121 It was reported to be a reversible, 2-OGcompetitive, pan-PHD inhibitor with IC50 values of 480, 280, and 450 nM for PHD 1, 2 and 3, respectively. Following a single oral administration of 70, EPO was significantly induced at doses of 1.25 mg/kg and above in male Wistar rats. In a 26day, repeat-dose study, treatment with 70 at 1.25 and 5 mg/kg doses increased PCV (packed cell volume) by 3% and 17%,

respectively, from baseline. The efficacy of 70 was compared to rhEPO in cynomolgus monkeys. A single dose of rhEPO (100 IU/kg, subcutaneous) or 70 (1.5 mg/kg, oral) increased hemoglobin and RBC to a similar extent in a two week study. Treatment with 70 showed an erythropoietic effect in different models of anemia in the rat. In a disease model of subtotal nephrectomy in rats, treatment with 70 led to a sustained reduction in mean systolic blood pressure. Thus, 70 demonstrated a better cardiovascular safety profile over rhEPO for the treatment of anemia associated with CKD. Compounds with a pyrazole central core (similar to Bayer compounds) described by Takeda are exemplified by 71−72.122 The essential rings such as 3-pyridyl and triazolyl with an N atom at the third position are absent in these molecules. The profiles of 434 compounds disclosed in this patent suggested the 4-pyridyl and 4-cyanophenyl rings as possible substitutes. The 2-pyridyl ring attached to the central core is attached to various secondary and tertiary amides. The compounds showed PHD inhibition in the range of pIC50 values spanning a range of 5.43−8.67, with most of the modulation achieved by exploring amines at the amide group. A cell-based HIF-stabilization assay was used to assess the PHD inhibitory activity of compounds. pIC50 values for 71 and 72 in the enzyme inhibition assay were found to be 8.4 and 8.0, respectively, while pEC50 values in the cell-based test were found to be 6.45 and 6.25, respectively. Compound 72 was also evaluated in the in vivo cardioprotection assay, at 5 mg/kg, 10 mg/kg, and 30 mg/kg in preclinical models. Compound 72 reduced the area of infarct in mice by 59% at 30 mg/kg and by 50% at 10 mg/kg compared to vehicle. Compound 72 also decreased markers of cardiac injury; the corresponding reduction of lactate dehydrogenase (LDH) released to the coronary effluent was 56% and 51% at 30 and 10 mg/kg, respectively. In mice, 72 at 60 mg/kg in C57BL/6 mice increased VEGF mRNA by 2-fold, when compared to 72, which could be responsible for protection against cardiac injury.122,123 Daiichi also reported another modification of the pyrazole class of compounds in the form of a novel series of EPO secretagogues exemplified by 73.124 The triazole and pyridine rings reported in 93−95 were replaced with an acetylamino group, as shown in 73. EPO induction activity of 27 examples was evaluated using Hep3b cell line. Compound 73 caused a 40-fold increase in EPO in this assay. Taisho has reported a series of triazole substituted heteroaryl amides exemplified by 74.125 The report of these molecules contains more than 200 examples with the data for PHD2 inhibition at 1 μM. One may postulate that the N atom from the triazole ring could form an iron chelating moiety with the adjacent 2-pyridyl ring. The free NH group may be involved in a H-bond interaction with Tyr303 similar to that reported for benzimidazole-based inhibitors 11 and 12. The IC50 value for 74 was 61 nM. The presence of an amide or reverse amide was preferred for high potency. However, any change such as an alkyl ether or an amide with an alkyl spacer at that position was found to be detrimental to activity. Compounds with an electron-releasing methoxy group para to the nitrogen atom of the pyridine ring have been found to be potent and effective. CrystalGenomics, Inc. has disclosed a series of phenolic compounds (e.g., 75 and 76).126 This group has also published the SAR and binding mode for a series of thiazole derivatives targeted at the 2-OG binding site.127 The presence of a cyano group was found to be the key to achieve PHD2 inhibitory potency during SAR evaluation of their initial hit. Compound 75 has shown the highest PHD2 inhibitory activity with an IC50 6974



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Figure 10. PHD inhibitors with the nonconventional binding mode.

value of 0.4 μM in an enzymatic assay. However, it did not show significant EPO release even at 100 μM in Hep3B cells due to its poor permeability in a parallel artificial membrane permeability assay (PAMPA). Compound 76 inhibited PHD2 with an IC50 of 2.1 μM, but it has shown around 40-fold EPO secretion at 50 μM concentration, which correlates with improved cell permeability. The cocrystal structure of an analogue related to 76 (PDB 4KBZ) suggests that it binds to Fe through the nitrogen atoms in the hydroxylpyridine and thiazole rings. This binding mode also enables the formation of an H-bond between the hydroxyl moiety and the conserved Tyr303. These compounds were potent due to an interaction of the cyano group with Tyr329 and Arg383, which is similar to the one shown by the carboxylic group-containing PHD inhibitors.

showed modest oral exposure (176 ng.h/mL) and high clearance (108 mL/min/kg). Brain drug levels were comparable to the plasma exposure (brain/plasma ratio 0.95), indicating the utility of the compound for the treatment of brain (CNS) related disorders. Compound 78 (IC50 = 0.074 μM) with a meta-methyl group in the top phenyl ring of 77 improved oral exposure (1869 ng·h/mL) while maintaining good CNS penetration (brain/plasma ratio 0.88). Compound 79 (IC50 = 0.09 μM), with an amino group introduced into the central core of 78, exhibited excellent peripheral exposure (40038 ng.h/mL) but poor CNS penetration (brain/plasma ratio 0.21). Compound 78 showed high protein binding, low solubility, and poor permeability, which are responsible for the poor penetration of the brain. Compound 78 was found to be a substrate for multidrug resistance protein (MDR)-1 in an in vitro MDCK-MDR-1 assay (efflux ratio 12). A multicentered team from the University of Oxford and the BioNanotechnology Research Centre from the Republic of Korea has published a series of diacylhydrazines as dual-action inhibitors of PHD. These PHD inhibitors bind to the active site iron. Also, these compounds bind with the cellular iron in a nonspecific way. Because diacylhydrazines possess considerable metal ion chelating ability and vulnerability to undergo oxidation to diacylazo-Michael acceptors, they can be toxic to the mammalian system.130 A broad profiling approach utilizing ESI-MS analysis of mixtures of the catalytic domain of PHD2, ferrous sulfate, and diacyl hydrazines (derived from various pyridine, quinolone, and isoquinoline carboxylic acids, hydrazine, and aliphatic diacids) enabled the identification of several analogues that bind this additional iron atom. Compound 80 inhibited PHD2 with an IC50 of 18.4 μM and showed increased HIF-1α levels in Hep3B cells. The Korea Institute of Science and Technology reported pyrithione Zn (81) as a highly selective PHD3 inhibitor by screening 1040 FDA-approved drugs and bioactive compounds.131 Pyrithione Zn is accepted worldwide as a microbicidal agent. It has shown IC50 value for PHD3 0.98 μM, while for PHD2 it was >1 mM. The contribution of Zn2+ to PHD3 inhibition was found to be nonsignificant. A mechanistic investigation led to the understanding that it is an allosteric inhibitor of PHD3.

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PHD INHIBITORS WITH NONCONVENTIONAL BINDING MODE Takeda has published a report on the bicyclic heteroaryl derivatives exemplified by 77−79 (Figure 10), which contains IC50 values of PHD1 inhibition for 262 examples.128 A recent publication also described SAR of compounds in this class.129 Modification of a lead identified from fragment screening campaign resulted in the identification of compound 77 (IC50 value of 0.034 μM). Both nonbridgehead nitrogen atoms of the 5-membered rings were found to be essential for PHD inhibition. More precisely, the nitrogen atom distant from the bridgehead nitrogen was found to be more important than the nitrogen atom next to it. The SAR showed a preference for electron withdrawing substituents in the 4-position of the phenyl ring with 4-CN group showing the best activity. Replacement of the phenyl ring with 5,6-membered heterocycles did not help to improve potency further. An X-ray cocrystal structure of compound 77 with PHD1 (PDB 5V1B) showed new monodentate coordination of the triazolopyridine N atom (away from the bridgehead nitrogen) with the active site Fe2+ ion, unlike the bidentate chelation interaction observed for almost all previously reported PHD inhibitors. The nitrile group showed a hydrogen bond interaction with the side chain of Asn315. The phenyl ring is positioned appropriately to make a π-stacking interaction with Tyr287, and the triazolopyridine N3 atom is potentially making a watermediated interaction with Tyr313. Compound 77, when evaluated for its PK profile in C57BL6 mice at 3 mg/kg, 6975



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LEARNINGS FROM THE CLINICAL TRIALS OF PROLYL HYDROXYLASE INHIBITORS The first rationally designed PHD inhibitor that reached clinical trials in ESRD patients for the treatment of anemia was 15. A modified analogue 16 (roxadustat) is currently in the late stage of phase III clinical trials. Earlier trials of 16 were carried out on patients with nondialysis dependent chronic kidney disease (ND-CKD) and dialysis-dependent CKD. In these trials, 16 improved anemia (defined as hemoglobin increase ≥1 g/dL) and maintained hemoglobin levels (defined as hemoglobin not falling more than 0.5 g/dL).39 It has also improved EPO release and decreased hepcidin in clinical trials, which can be considered the first proof of principle for PHD inhibitors in late-stage clinical trials without a significant safety concern. Compound 16 could be the first molecule in the PHD class to reach the clinic for the treatment of anemia associated with chronic kidney disease patients. It has a half-life of 12 h after oral dosing. When 16 was administered in doses of either 1.0 or 2.0 mg/kg twice weekly (BIW) or three times weekly (TIW), endogenous EPO release started within 4 h, which reached a peak at 10 h after dosing. The increase in EPO was reversible because endogenous EPO levels returned to baseline a day after administration. Maximum serum EPO levels after administration of 1 mg/kg roxadustat were 115 mU/mL, which was significantly lower than those achieved by intravenous injection of recombinant erythropoietin. After 6 weeks of treatment, 16 has shown a significant decrease in hepcidin levels, which has translated into reducing functional iron deficiency. After these initial trials, 16 has undergone clinical trials in more than 1000 patients.39 Apart from EPO, HIF activation causes VEGF stimulation that can lead to angiogenesis and induce tumor progression. However, despite the induction of erythropoiesis, 16 does not promote tumor initiation, progression, or metastasis in a VEGF-sensitive model of spontaneous breast cancer.132 Compound 16 is a pan-PHD inhibitor (which inhibits all the three PHD isoforms), and the dosing frequency was mostly biweekly or triweekly doses, ranging from 1 to 2 mg/kg. It is expected to enter the clinical use in late 2018. Compound 34 or daprodustat (GSK1278863) is currently undergoing phase III clinical trials.133 In a double-blind, placebo-controlled trial in anemia patients on hemodialysis (HD), 34 increased hemoglobin in a dose-related manner after 4 weeks of treatment. A dose-dependent increase in plasma EPO concentration was observed up to 8 mg dose, where the response was saturated. Serum EPO levels increased beyond 500 mIU/mL in the patients treated with 10 mg of 34, although VEGF did not change. However, daprodustat affects cardiac repolarization. Statistically significant decreases in the ΔQTc were observed by daprodustat treatment, along with higher incidences of gastrointestinal adverse events.134 Compound 23 (vadadustat/AKB-6548) is currently in phase III clinical trials. In phase I trials, 23 (900 mg) increased the peak serum concentration of EPO to 32.4 mU/mL at 18 h after treatment in healthy adults.135 In a phase II study, 23 treatment for 6 weeks has significantly increased hemoglobin in a dosedependent manner and also increased iron-binding capacity in the blood. It was associated with a decrease in ferritin and hepcidin concentration, indicating a significant improvement in iron homeostasis for efficient erythropoiesis. There were no changes in blood pressure, VEGF, C-reactive protein, or total cholesterol.135 A transient decrease in mean arterial blood

pressure and a mild transient increase in serum uric acid level was observed in a 4-week dose escalation study.136 Compound 70 (molidustat/BAY 85-3934) is a pan-PHD inhibitor, which is currently in phase II clinical trials. The dose of 5−50 mg of 95 has resulted in a dose-dependent increase in serum EPO levels, with a peak of 39.8 mU/mL, at 12 h after a single dose of 50 mg in healthy humans, when compared to 14.8 mU/mL for placebo.137 Compound 36 (desidustat/ZYAN1), is a pan-PHD inhibitor in phase II clinical trials. Preclinical profile of 36 indicates that it has a potential to treat anemia associated with CKD and chemotherapy-induced anemia at equivalent doses. It has demonstrated hematinic potential by combined effects on EPO release and efficient iron utilization owing to hepcidin suppression.138 Its pharmacokinetics is minimally affected in patients with CKD.139 In phase I clinical trials conducted in Australia and India, single (10−300 mg) and multiple doses (100−300 mg) of 36 in healthy subjects was found to be safe and well-tolerated. Administration of 36 was associated with a dose-related increase in C max and AUC. Serum EPO concentrations showed a trend of dose−response as well.93 On the basis of the t1/2, pharmacodynamic activity, and lack of drug accumulation, this compound is suitable for alternate day dosing in the clinic, which is currently in phase II clinical trials. There are a few other compounds like 54 (JTZ-951), JNJ42905343, and DS-1093. However, no significant development activities have been published for these compounds.

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CONCLUSION The discovery of prolyl hydroxylase enzyme inhibitors introduced new therapeutic options in the field of anemia associated with chronic diseases. More than a decade after the development of the first molecule that reached clinical trials, the understanding of prolyl hydroxylase inhibitors has increased extensively, with the conclusive evidence that inhibition of PHD could provide an exciting approach for the treatment of anemia associated with CKD. Many safe and effective PHD inhibitors followed the discovery of 15. These PHD inhibitors consist of a 2-oxoglutarate scaffold. These compounds blocked the interaction between 2-OG and prolyl hydroxylase and also stabilized the closed conformation of the PHD−inhibitor complex, which inhibited the substrate binding for degradation of HIF. Thus, carboxylic acid-based PHD inhibitors were discovered that incorporated an isoquinoline ring and which were optimized to the most advanced clinical candidate 16. Akebia Therapeutics has developed monocyclic pyridine derivative 23, GSK developed pyrimidine-trione derivative 34, and Cadila Healthcare developed N-alkoxyquinolone derivative 36, which maintains the carboxylic acid chain intact. Bayer took a different approach to the development of a noncarboxylic acid-based moiety, 70. Compound 70 achieved 2-fold iron chelation using the ring nitrogen atoms of the pyrazole core and the adjacent pyrimidine ring, with an added interaction of the triazole moiety with the 2-OG binding residue Arg-383. These compounds have been developed for a once daily or an alternate day (thrice a week) dosing regimen. The alternative day dosing regimen was designed to address the compliance issue in dialysis patients, where the drug administration needs to be in synchrony with a dialysis routine. On the other hand, an overly long half-life may invites safety issues due to prolonged stabilization of HIF. The Merck group’s optimization of 63 highlighted the importance of a structure−activity approach balancing potency, reducing side effects, and 6976



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achieving the desired half-life owing to increased metabolism of the compound. PHD inhibitors stimulate renal and hepatic erythropoiesis, along with the promotion of efficient iron utilization. They induce a transient increase in the expression of HIF-regulated genes, including renal and hepatic EPO, and maintain a physiological concentration of EPO in blood. The peak EPO concentration after a clinically useful dose of 16, 23, 34, 36, and 70 have resulted in EPO levels of 32.4−500 mU/mL in circulation, which is many-fold lower than those achieved by recombinant erythropoietin administration. Thus, PHD inhibitors are a safer therapeutic option for the treatment of anemia associated with CKD. An underestimated advantage of PHD inhibitors lies in their positive modulation of iron homeostasis under anemic conditions. CKD and other inflammatory disorders are associated with increased hepcidin. By suppression of hepcidin, PHD inhibitors improve functional iron deficiency, which may reduce the need for iron supplementation (Figure 11).

response from the kidneys. However, to achieve a significant erythropoiesis response from the liver (as in anephric patients), simultaneous inhibition of PHD1, PHD2, and PHD3 are necessary. On the other hand, HIF activation has a widespread effect on the body, which causes angiogenesis and oncogenesis due to vascular responses to hypoxia. Hence, PHD inhibitors may arouse a safety concern in situations like diabetic nephropathy or malignancies. However, the induction of erythropoiesis by PHD inhibition has been reported not to increase tumor progression in VEGF-sensitive models of cancer. If translated to humans, this phenomenon may be useful to position PHD inhibitors for the treatment of chemotherapy-induced anemia in cancer patients. The majority of the PHD inhibitors were designed based on a 2-OG mimetic approach using in vitro and in vivo screening, although some have been discovered by an HTS approach. The in vitro assays used for the testing of compounds are not uniform across the laboratories, mostly due to the use of different active fragment proteins of HIF and PHD. On the other hand, the in vivo screening using hematocrit as the marker of erythropoiesis has successfully translated to the clinic for most of the clinically advanced compounds. HIF signaling is a complicated process which affects many vital pathways including glucose and lipid metabolism, angiogenesis, and vascular metabolism. The role of specific PHD isoforms or HIF-α isoforms in the regulation of these different pathways is not yet adequately delineated. Hence, it is difficult to distinguish the toxicities of PHD inhibitors that are related to the mechanism and those which are off-target. Still, PHD inhibitors are safe in CKD patients. However, the effect in longterm clinical studies using the markers of tumor progression, angiogenesis, and cardio-metabolic abnormalities are necessary in order to expand the therapeutic use of PHD inhibitors to condition like chemotherapy-induced anemia and anemia associated with aging.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +91-2717-665555. Fax: +91-2717-665551. E-mail: [email protected].

Figure 11. Mechanism of action of HIF-PHD inhibitors. The chronic inflammatory condition or kidney disease increases the release of inflammatory cytokines and decreases erythropoietin release. Inflammatory cytokines stimulate hepcidin synthesis. Increased levels of hepcidin downregulate DMT1, Dcytb, and block ferroportin and inhibit iron absorption. Hepcidin degrades ferroportin in macrophages and the liver, which leads to reduced iron availability in the blood and increases intracellular iron. The decrease in EPO and increased hepcidin inhibit hemoglobin synthesis and the maturation of RBCs, resulting in anemia. PHD inhibitors stabilize HIF and stimulate EPO release and decrease hepcidin levels and thus stimulate efficient erythropoiesis.

ORCID

Amit A. Joharapurkar: 0000-0003-0723-4371 Vrajesh B. Pandya: 0000-0001-9004-4514 Notes

The authors declare the following competing financial interest(s): All the authors are employee(s) or consultant(s) of Zydus Research Centre, a unit of Cadila Healthcare Limited, which develops PHD inhibitors. The authors have no other conflicts of interest to report. Biographies Amit A. Joharapurkar received his Ph.D. in 2005, after which he followed a career in research at the Zydus Research Centre, Ahmedabad, India, where he has led pharmacology efforts for several programs in the metabolic disorders therapeutic area, including the discovery of PHD inhibitors. He is the recipient of the CDRI Award for Excellence in Drug Research 2018. His scientific interests include obesity, diabetes, dyslipidemia, and anemia.

Loss of function mutations in humans and PHD gene knockout data in mice have indicated that inhibition of PHDs can be useful in some indications related to hypoxia and vascular responses, although diseases other than anemia associated with CKD have yet to establish clinical proof of concept. Studies in PHD knockout mice suggest that the effect of PHD inhibitors is dependent on the extent of activation of the different isoforms of PHDs (PHD1, 2, and 3). It also depends on how the erythropoiesis in the kidney and the liver are differentially affected. On the other hand, PHD2 inhibition alone is enough to deliver a clinically relevant erythropoiesis

Vrajesh B. Pandya is a medicinal chemist at Zydus Research Centre, Cadila Healthcare Limited, Ahmedabad, India. He obtained his Ph.D. degree from The Maharaja Sayajirao University of Baroda, Vadodara, India. His work in medicinal chemistry focused on novel compounds 6977



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receptor-1; IC50, half-maximal inhibitory concentration; EC50, half-maximal effective concentration; CP4H, collagen prolyl hydroxylase; FIH, factor inhibiting HIF; HTS, high throughput screening; HTRF, homogeneous time resolved fluorescence; hERG, human ether-a-go-go-related gene; PK, pharmacokinetic; PCV, packed cell volume; rhEPO, human recombinant erythropoietin; LDH, lactate dehydrogenase; SAR, structure− activity relationship; PAMPA, parallel artificial membrane permeability assay; CNS, central nervous system; MDR, multidrug resistance protein; FDA, U.S. Food and Drug Administration; BIW, twice weekly; TIW, three times weekly; ESRD, end stage renal disease; ND-CKD, nondialysis dependent chronic kidney disease; HD, hemodialysis

for the treatment of thrombotic and related disorders. He has contributed to the discovery and development of PHD inhibitors at Zydus Research Centre. Vishal J. Patel received his Ph.D. in Pharmacology from Kadi Sarva Vishwavidyalaya, India. His Ph.D. project focused on the development of coagonist of glucagon-derived peptide hormones as useful tools in the treatment of metabolic disorders. He has worked as a pharmacologist in Torrent Research Centre, India, and joined Zydus Research Centre in 2007. In Zydus Research Centre, he is involved in screening and development of new chemical entities for obesity, dyslipidemia, and anemia programs. He is the recipient of WileyJournal of Diabetes Young Investigator Award in 2013, and Keystone Symposia Future of Science Fund scholarship in 2016,

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Ranjit C. Desai was a Senior Vice President and Head of Chemistry (July 2012−Dec 2017) at Zydus Research Centre, Cadila Healthcare Ltd, Ahmedabad, India. He is the inventor of desidustat, a PHD inhibitor currently in phase 2 clinical trials. Dr. Desai received his Ph.D. from the MS University of Baroda, India, working with Dr. Sukh Dev at the Malti-Chem Research Centre. After working as a postdoctoral associate at Clemson University, Purdue University, and the University of Montreal, he worked at Sanofi-Winthrop, Hoechst Celanese, and Merck Research Laboratories. Currently, he is a Senior Consultant at Zydus Research Centre and runs projects in the metabolic diseases and antibacterial area.

REFERENCES

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Mukul R. Jain is President at Zydus Research Centre, Cadila Healthcare Ltd., Ahmedabad, India. His team is involved in discovery and development of new drugs. Dr. Jain obtained his M.Pharm. and Ph.D. from Nagpur University, India. After completing Ph.D., he worked at Wockhardt and Ranbaxy Research Centers before moving to the University of Florida at Gainesville as a Postdoc Associate. He then worked as Assistant Professor in the National Institute of Pharmaceutical Education and Research, Mohali, India, before joining Zydus Research Centre in 2000. His group at Zydus Research Centre has developed several NCEs including Saroglitazar and Desidustat and also contributed to the development of several biologics & vaccines of Cadila Healthcare Ltd.

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ACKNOWLEDGMENTS The authors are grateful to Mr. Pankaj R. Patel, Chairman of Cadila Healthcare Ltd., India for his guidance & support.

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ABBREVIATIONS USED AUC, area under the curve; EPO, erythropoietin; ESA, erythrocyte-stimulating agents; PHD, prolyl hydroxylase domain; HIF, hypoxia-inducible transcription factor; RBC, red blood cells; CKD, chronic kidney disease; HSC, hematopoietic stem cells; CFU-E, colony forming units, erythroid; EPOR, EPO receptors; rhEPO, human recombinant EPO; JAK2, Janus tyrosine kinase-2; STAT, signal transduction and activator of transcription; AKT kinase, protein kinase B; CD95, cluster of differentiation 95; TNF, tumor necrosis factor; FPN, ferroportin; BMP/SMAD, bone morphogenetic protein/ SMAD; IL-6, interleukin 6; ERFE, erythroferrone; pVHL, Hippel−Lindau tumor suppressor protein; 2-OG, 2-oxoglutarate; TRF, transferrin; DMT1, divalent metal transporter 1; Dcytb, duodenal cytochrome b; TRFR, transferrin receptor; VEGF, vascular endothelial growth factor; NOG, N-oxalylglycine; DMOG, dimethyloxalylglycine; IBD, inflammatory bowel disease; GM-CSF, granulocyte-macrophage colony-stimulating factor; VEGFR-1, vascular endothelial growth factor receptor-1; sVEGFR-1, suppressor of vascular endothelial growth factor 6978



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